Augmented cognitive training

ABSTRACT

The present invention provides methods of treating cognitive deficits associated with mental retardation. The methods comprise combining cognitive training protocols and a general administration of phosphodiesterase 4 inhibitors.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/927,914, filed on Aug. 10, 2001, which claims the benefit ofU.S. Provisional Application No. 60/224,227, filed on Aug. 10, 2000. Theentire teachings of these application are incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] An estimated 4 to 5 million Americans (about 2% of all ages and15% of those older than age 65) have some form and degree of cognitivefailure. Cognitive failure (dysfunction or loss of cognitive functions,the process by which knowledge is acquired, retained and used) commonlyoccurs in association with central nervous system (CNS) disorders orconditions, including age-associated memory impairment, delirium(sometimes called acute confusional state), dementia (sometimesclassified as Alzheimer's or non-Alzheimer's type), Alzheimer's disease,Parkinson's disease, Huntington's disease (chorea), mental retardation,cerebrovascular disease (e.g., stroke, ischemia), affective disorders(e.g., depression), psychotic disorders (e.g., schizophrenia, autism(Kanner's Syndrome)), neurotic disorders (e.g., anxiety,obsessive-compulsive disorder), attention deficit disorder (ADD),subdural hematoma, normal-pressure hydrocephalus, brain tumor, head orbrain trauma.

[0003] Cognitive dysfunction is typically manifested by one or morecognitive deficits, which include memory impairment (impaired ability tolearn new information or to recall previously learned information),aphasia (language/speech disturbance), apraxia (impaired ability tocarry out motor activities despite intact motor function), agnosia(failure to recognize or identify objects despite intact sensoryfunction), disturbance in executive functioning (i.e., planning,organizing, sequencing, abstracting).

[0004] Cognitive dysfunction causes significant impairment of socialand/or occupational functioning, which can interfere with the ability ofan individual to perform activities of daily living and greatly impactthe autonomy and quality of life of the individual.

[0005] Cognitive training protocols are generally employed inrehabilitating individuals who have some form and degree of cognitivedysfunction. For example, cognitive training protocols are commonlyemployed in stroke rehabilitation and in age-related memory lossrehabilitation. Because multiple training sessions are often requiredbefore an improvement or enhancement of a specific aspect of cognitiveperformance (ability or function) is obtained in the individuals,cognitive training protocols are often very costly and time-consuming.

SUMMARY OF THE INVENTION

[0006] The present invention relates to a novel methodology, alsoreferred to herein as augmented cognitive training (ACT), which caneither (1) rehabilitate various forms of cognitive dysfunction moreefficiently than any current method or (2) enhance normal cognitiveperformance (ability or function). ACT can be applied for any aspect ofbrain function that shows a lasting performance gain after cognitivetraining. Accordingly, ACT can be used in rehabilitating an animal withsome form and degree of cognitive dysfunction or in enhancing(improving) normal cognitive performance in an animal. ACT can also beused to exercise appropriate neuronal circuits to fine-tune the synapticconnections of newly acquired, transplanted stem cells thatdifferentiate into neurons.

[0007] As described herein, ACT comprises two indivisible parts: (1) aspecific training protocol for each brain (cognitive) function and (2)administration of cyclic AMP response element binding protein (CREB)pathway-enhancing drugs. This combination can augment cognitive trainingby reducing the number of training sessions required to yield aperformance gain relative to that obtained with cognitive training aloneor by requiring shorter or no rest intervals between training sessionsto yield a performance gain. This combination can also augment cognitivetraining by reducing the duration and/or number of training sessionsrequired for the induction in a specific neuronal circuit(s) of apattern of neuronal activity or by reducing the duration and/or numberof training sessions or underlying pattern of neuronal activity requiredto induce CREB-dependent long-term structural/function (i.e.,long-lasting) change among synaptic connections of the neuronal circuit.In this manner, ACT can improve the efficiency of existing cognitivetraining protocols, thereby yielding significant economic benefit.

[0008] For example, cognitive training protocols are employed intreating patients with depression (monopolor) and/or phobias to helpthem unlearn pathological responses associated with the depressionand/or phobia(s) and learn appropriate behavior. Administration of aCREB pathway-enhancing drug in conjunction with cognitive trainingreduces the time and/or number of training sessions required to yield again in performance in these patients. As such, overall treatment isaccomplished in a shorter period of time.

[0009] Similarly, cognitive training protocols are employed in treatingpatients with autism to help them unlearn pathological responses and tolearn appropriate behavior. Administration of a CREB pathway-enhancingdrug in conjunction with cognitive training reduces the time and/ornumber of training sessions required to yield a gain in performance inthese patients.

[0010] Cognitive training protocols (e.g., physical therapy,bio-feedback methods) are employed in rehabilitating stroke patients(stroke rehabilitation), particularly rehabilitating impaired or lostsensory-motor function(s). Administration of a CREB pathway-enhancingdrug in conjunction with cognitive training reduces the time and/ornumber of training sessions required to yield a gain in performance inthese patients. Faster and more efficient recovery of lost cognitivefunction(s) are expected as a result.

[0011] Cognitive training protocols (e.g., massed training, spacedtraining) are employed in learning a new language or in learning to playa new musical instrument. Administration of a CREB pathway-enhancingdrug in conjunction with cognitive training reduces the time and/ornumber of training sessions required to yield a gain in performance. Asa result, less practice (training sessions) is required to learn the newlanguage or to learn to play the new musical instrument.

[0012] Cognitive training protocols are employed in improving learningand/or performance in individuals with learning, language or readingdisabilities. Administration of a CREB pathway-enhancing drug inconjunction with cognitive training reduces the time and/or number oftraining sessions required to yield a gain in performance in theseindividuals.

[0013] Cognitive training protocols are employed to exercise neuronalcircuits in individuals to fine-tune synaptic connections of newlyacquired, transplanted stem cells that differentiate into neurons.Administration of a CREB pathway-enhancing drug in conjunction withcognitive training reduces the time and/or number of training sessionsrequired for the induction in (a) specific neuronal circuit(s) of apattern of neuronal activity in these individuals.

[0014] Cognitive training protocols are employed for repeatedstimulation of neuronal activity or a pattern of neuronal activityunderlying (a) specific neuronal circuit(s) in individuals.Administration of a CREB pathway-enhancing drug in conjunction withcognitive training reduces the time and/or number of training sessionsand/or underlying pattern of neuronal activity required to induceCREB-dependent long-term structure/function (i.e., long-lasting) changeamong synaptic connections of the neuronal circuit.

[0015] As a result of the present invention, methods of enhancing aspecific aspect of cognitive performance in an animal (particularly ahuman or other mammal or vertebrate) in need thereof are provided hereincomprising (a) administering to the animal an augmenting agent whichenhances CREB pathway function; and (b) training the animal underconditions sufficient to produce an improvement in performance of acognitive task of interest by the animal. “Augmenting agents” are alsoreferred to herein as “CREB pathway-enhancing drugs”.

[0016] Methods are provided herein for treating a cognitive deficitassociated with a central nervous system (CNS) disorder or condition inan animal in need of said treatment comprising (a) administering to theanimal an augmenting agent which enhances CREB pathway function; and (b)training the animal under conditions sufficient to produce animprovement in performance of a particular cognitive task by the animal.CNS disorders and conditions include age-associated memory impairment,neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson'sdisease, Huntington's disease (chorea), other senile dementia),psychiatric diseases (e.g., depression, schizophrenia, autism, attentiondeficit disorder), trauma dependent loss of function (e.g.,cerebrovascular diseases (e.g., stroke, ischemia), brain tumor, head orbrain injury), genetic defects (e.g., Rubinstein-Taybi syndrome, downsyndrome, Angelman syndrome, neurofibromatosis, Coffin-Lowry syndrome,Rett syndrome, myotonic dystrophy, fragile X syndrome (e.g., fragileX-1, fragile X-2), William's syndrome) and learning disabilities.

[0017] In a particular embodiment, methods are provided herein fortreating a cognitive deficit associated with mental retardation in ananimal in need of said treatment comprising (a) administering to theanimal an augmenting agent which enhances CREB pathway function (e.g., aphosphodiesterase 4 inhibitor); and (b) training the animal underconditions sufficient to produce an improvement in performance by theanimal of a cognitive task whose deficit is associated with mentalretardation. Mental retardation impacts cognitive processing andcognitive functions, including learning and memory acquisition. Mentalretardation may be caused by chromosomal or genetic factors, congenitalinfections, teratogens (drugs and other chemicals), malnutrition,radiation or unknown conditions affecting implantation andembryogenesis. Mental retardation syndromes include Rubinstein-Taybisyndrome, down syndrome, Angelman syndrome, neurofibromatosis,Coffin-Lowry syndrome, Rett syndrome, myotonic dystrophy, fragile Xsyndrome (e.g., fragile X-1, fragile X-2) and William's syndrome(Weeber, E. J. et al., Neuron, 33:845-848 (2002)).

[0018] Methods are also provided herein for therapy of a cognitivedeficit associated with a CNS disorder or condition in an animal havingundergone neuronal stem cell manipulation comprising (a) administeringto the animal an augmenting agent which enhances CREB pathway function;and (b) training the animal under conditions sufficient to stimulate orinduce neuronal activity or a pattern of neuronal activity in theanimal. By “neuronal stem cell manipulation” is meant that (1) exogenousneuronal stem cells are transplanted into the brain or spinal chord ofan animal or (2) endogenous neuronal stem cells are stimulated orinduced to proliferate in the animal.

[0019] Methods are provided herein for repeated stimulation of neuronalactivity or a pattern of neuronal activity, such as that underlying aspecific neuronal circuit(s), in an animal comprising (a) administeringto the animal an augmenting agent which enhances CREB pathway function;and (b) training the animal under conditions sufficient to stimulate orinduce neuronal activity or a pattern of neuronal activity in theanimal.

BRIEF DESCRIPTION OF THE DRAWING

[0020]FIG. 1 is a schematic diagram illustrating a neuronal mechanism ofbrain plasticity, which forms the neurological basis for augmentedcognitive training. Specific cognitive training protocols produce(experience-dependent) changes in neural activity of specific underlyingneuronal circuits. This neural activity activates a biochemical processthat modulates CREB-dependent gene expression. Downstream effectors ofthis transcription factor cascade then yield long-lasting structural andfunctional changes in synaptic connectivity of the circuit (i.e.,long-term memory) (Dubnau J. et al., Current Biology, 13: 286-296(2003)). This process of experience-dependent synaptic modification isongoing in normal animals and usually requires multiple trainingsessions for most tasks. Augmentation of the CREB pathway duringtraining will reduce the number of training sessions (or shorten therest interval between them) required to produce the experience-dependentchanges in synaptic structure and/or function.

[0021]FIG. 2A is a bar graph representation depicting results showingthat the PDE4 inhibitors rolipram and HT0712 enhance forskolin-inducedgene expression in human neuroblastoma cells. Relative light units (RLU)emitted from luciferase were quantified in human neuroblastoma cellsstably transfected with a CRE-luciferase reporter gene and exposed tovehicle alone or drug (HT0712 or rolipram) for two hours beforestimulation by a suboptimal dose of forskolin. The results show thatboth drugs increased forskolin-induced CRE-luciferase expression1.9-fold above forskolin alone when assayed 4 hours after forskolinstimulation.

[0022]FIG. 2B is a bar graph representation depicting results showingthat the PDE4 inhibitors rolipram and HT0712 enhance forskolin-inducedgene expression in human neuroblastoma cells. Real-time PCR was used toquantify expression of somatostatin, an endogenous cAMP-responsive gene.Expression levels induced by forskolin or by forskolin+drug arequantified as differences in threshold cycle number (ΔC₁) above vehiclealone control groups. The results show that HT0712 and rolipram produced4.6-fold and 2.3-fold increases in forskolin-induced expression ofsomatostatin, respectively.

[0023]FIG. 3 is a bar graph representation depicting results showingthat CBP³⁵ mice have impaired long term memory in object recognitiontask. Wildtype mice and CBP^(±) mutant mice were trained for 15 minutesand tested 3 hours or 24 hours later. Memory retention was quantified asa Discrimination Index, the fraction of time spent exploring a novelversus a familiar object. Three-hour memory levels were similar fornormal mice and CBP^(±) mutants (p=0.76; n=6 for each genotype), but 24hour memory was significantly lower than normal in mutant mice (p<0.01;n=10 for each genotype).

[0024]FIG. 4 is a bar graph representation depicting results showingthat the PDE4 inhibitors HT0712 and rolipram ameliorate the long-termmemory defect in CBP^(±) mutant mice. Wildtype mice and CBP^(±) mutantmice received 0.1 mg/kg HT 0712 or rolipram injected i.p. 20 minutesbefore training. Animals experienced a 15 minute training session andmemory retention was tested 24 hours later. In vehicle-injected CBP^(±)mutants, memory was significantly lower than in vehicle-injectedwildtype mice (p<0.01; n=12 and n=6, respectively). In drug-injectedCBP^(±) mutants, memory was significantly higher than invehicle-injected mutants (p<0.05; N=10 and N=12, respectively, forHT0712; p<0.05, N=8 and N=2, respectively, for rolipram). Memoryretention did not differ significantly between drug-treated CBP^(±)mutants and drug-treated wildtype mice (p=0.78, N=10 for each group forHT0712; p=0.19, N=8 and N=10, respectively, for rolipram).

[0025]FIG. 5 is a bar graph representation depicting results showing adose response curve for HT0712 in wildtype mice. Mice received a singlei.p. injection of drug or vehicle alone 20 minutes before training.Doses of 0.001 mg/kg, 0.005 mg/kg, 0.01 mg/kg, 0.05 mg/kg, 0.10 mg/kg,0.15 mg/kg, 0.20 mg/kg and 0.50 mg/kg were used. Animals experienced a3.5 minute training protocol and were tested 24 hours later. Memoryretention in drug-injected animals was significantly higher than that invehicle-alone animals (N=35) at doses of 0.05 mg/kg (N=20; p<0.05), 0.10mg/kg (N=22;p<0.0001)and 0.15 mg/kg(N=18;p<0.001).

[0026]FIG. 6 is a graphical representation depicting results showingthat CBP^(±) mutants and wildtype mice show a different dose sensitivityto HT0712. CBP^(±) mutants and wildtype mice received a single i.p.injection of vehicle or drug 20 minutes before training. Theyexperienced a 3.5 minute training protocol and were tested 24 hourslater. In wildtype mice, memory retention in drug-treated groups washigher than in the vehicle-alone group (N=26) at doses of 0.05 mg/kg(N=12; p<0.005), 0.10 mg/kg (N=8; p<0.0001), 0.15 mg/kg (N=18; p<0.005)and 0.2 mg/kg (N=14; p<0.00 In CBP^(±) mutants, memory retention indrug-treated groups was higher than in the vehicle along group (N=26) atdoses of 0.10 mg/kg (N=8; p<0.0001), 0.15 mg/kg (N=10; p<0.0001) and 0.2mg/kg (N=14; p<0.0001). In contrast to wildtype mice, a 0.05 mg/kg doseof HT0712 failed to enhance memory in CBP^(±) mutants (N=26; p=0.79).

DETAILED DESCRIPTION OF THE INVENTION

[0027] For many tasks in many species, including human, spaced trainingprotocols (multiple training sessions with a rest interval between each)produce stronger, longer-lasting memory than massed training protocols(multiple training sessions with no rest interval in between).Behavior-genetic studies of Pavlovian olfactory learning in Drosophilahave established that massed training produces a long-lasting memorythat nevertheless decays away in at least four days, is not proteinsynthesis-dependent, is not disrupted by overexpression of aCREB-repressor transgene, and is disrupted in radish mutants (Tully, T.et al., Cell, 79(1):35-47 (1994); and Yin, J. C. et al., Cell,79(l):49-58 (1994)). In contrast, spaced training produces along-lasting memory that persists for at least seven days, is proteinsynthesis-dependent, is disrupted by overexpression of a CREB-repressortransgene and is normal in radish mutants (Tully, T. et al., Cell,79(1):35-47 (1994); and Yin, J. C. et al., Cell, 79(1):49-58 (1994)).One day after spaced training, memory retention is composed of both theprotein synthesis- and CREB-independent early memory (ARM) and theprotein synthesis- and CREB-dependent long-term memory (LTM). Additionalmassed training is insufficient to induce LTM (Tully, T. et al., Cell,79(l):35-47 (1994); and Yin, J. C. et al., Cell, 79(1):49-58 (1994)).

[0028] A growing body of evidence extends these results frominvertebrates to mammals. For example, in Aplysia, molecularmanipulations of CREB expression, similar to those in flies, suppress orenhance (i) LTM of a facilitatory electrophysiological response at asensorimotor monosynapse in cell culture and (ii) the synapticconnections between sensory and motor neurons that are normally producedafter spaced applications of the facilitatory stimulus (Bartsch, D. etal., Cell, 83(6):979-992 (1995)). In rats, injections of antisense RNAoligonucleotides into hippocampus or amygdala block LTM formation of twodifferent tasks that are dependent on activity in these anatomicalregions, respectively (Guzowski, J. F. et al., Proc. Natl. Acad. Sci.USA, 94(6):2693-2698 (1997); and Lamprecht, R. et al., J. Neurosci.,17(21):8443-8450 (1997)). In mice, LTM formation for both implicit andexplicit tasks is defective in CREB mutant mice (Bourtchuladze, R. etal., Cell, 79(1):59-68 (1994)).

[0029] Training of transgenic mice, carrying a CRE-dependent reportergene (beta-galactosidase), in hippocampal-dependent contextual fearconditioning or passive avoidance tasks induces CRE-dependent reportergene expression in areas CA1 and CA3 of the hippocampus. Training ofthese mice in an amygdala-dependent fear conditioning task inducesCRE-dependent reporter gene expression in the amygdala, but not thehippocampus. Thus, training protocols that induce LTM formation alsoinduce CRE-dependent gene transcription in specific anatomical areas ofthe mammalian brain (Impey, S. et al., Nat. Neurosci., 1(7):595-601(1998)).

[0030] With these animal models, three salient cases of LTM enhancementhave been demonstrated. First, overexpression of a CREB-activatortransgene abrogates the requirements for multiple, spaced trainingsessions and, instead, induces LTM formation after only one trainingsession (which normally produces little or no memory retention 24 hourslater (Yin, J. C. et al., Cell, 81(1):107-115 (1995)). Second, injectionof a virally expressed CREB-activator transgene into rat amygdala alsois sufficient to enhance memory after massed training for thefear-potentiated startle response, which abrogates the requirement for arest interval in spaced training (Josselyn, S. A. et al., Society forNeuroscience, Vol. 24, Abstract 365.10 (1998); and Josselyn, S. A. etal., J. Neurosci., 21:2404-2412 (2001)). Third, LTM formation inCREB-deficient mice (Bourtchuladze, R. et al., Cell, 79(1):59-68 (1994))can form normally, if mutant mice are subjected to a different, spacedtraining protocol (Kogan, J. H. et al., Curr. Biol., 7(1):1-11 (1997)).

[0031] CREB also appears involved in various forms of developmental andcellular plasticity in the vertebrate brain. For example, neuronalactivity increases CREB activity in the cortex (Moore, A. N. et al., J.Biol. Chem., 271(24):14214-14220 (1996)). CREB also mediatesdevelopmental plasticity in the hippocampus (Murphy, D. D. et al., Proc.Natl. Acad. Sci. USA, 94(4):1482-1487 (1997)), in the somatosensorycortex (Glazewski, S. et al., Cereb. Cortex, 9(3):249-256 (1999)), inthe striatum (Liu, F. C. et al., Neuron, 17(6):1133-1144 (1996)), and inthe visual cortex (Pham, T. A. et al., Neuron, 22(1):63-72 (1999)).

[0032] CREB appears to be affected in human neurodegenerative diseaseand brain injury. For example, CREB activation and/or expression isdisrupted in Alzheimer's disease (Ikezu, T. et al., EMBO J.,15(10):2468-2475 (1996); Sato, N. et al., Biochem. Biophys. Res.Commun., 232(3):637-642 (1997); Yamamoto-Sasaki, M. et al., Brain. Res.,824(2):300-303 (1999); Vitolo, O. V. et al., Proc. Natl. Acad. Sci. USA,13217-13221 (2002)). CREB activation and/or expression is also elevatedafter seizures or ischemia (Blendy, J. A. et al., Brain Res.,681(1-2):8-14 (1995); and Tanaka, K. et al., Neuroreport, 10(11):2245-2250 (1999)). “Environmental enrichment” is neuroprotective,preventing cell death by acting through CREB (Young, D. et al., Nat.Med., 5(4):448-453 (1999)).

[0033] CREB functions during drug sensitivity and withdrawal. Forexample, CREB is affected by ethanol (Pandey, S. C. et al., AlcoholClin. Exp. Res., 23(9):1425-1434 (1999); Constantinescu, A. et al., J.Biol. Chem., 274(38):26985-26991 (1999); Yang, X. et al., Alcohol Clin.Exp. Res., 22(2):382-390 (1998); Yang, X. et al., J. Neurochem.,70(1):224-232 (1998); and Moore, M. S. et al., Cell, 93(6):997-1007(1998)), by cocaine (Carlezon, W. A., Jr. et al., Science,282(5397):2272-2275 (1998)), by morphine (Widnell, K. L. et al., J.Pharmacol. Exp. Ther., 276(1):306-315 (1996)), by methamphetamine(Muratake, T. et al., Ann N.Y. Acad. Sci., 844:21-26 (1998)) and bycannabinoid (Calandra, B. et al., Eur. J. Pharmacol., 374(3):445-455(1999); and Herring, A. C. et al., Biochem. Pharmacol., 55(7): 1013-1023(1998)).

[0034] A signal transduction pathway that can stimulate the CREB/CREtranscriptional pathway is the cAMP regulatory system. Consistent withthis, mice lacking both adenylate cyclase 1 (AC1) and AC8 enzymes failto learn (Wong S. T. et al., Neuron, 23(4):787-798 (1999)). In thesemice, administration of forskolin to area CA1 of the hippocampusrestores learning and memory of hippocampal-dependent tasks.Furthermore, treatment of aged rats with drugs that elevate cAMP levels(such as rolipram and D1 receptor agonists) ameliorates an age-dependentloss of hippocampal-dependent memory and cellular long-term potentiation(Barad, M. et al., Proc. Natl. Acad. Sci. USA, 95(25):15020-15025(1998)). These latter data suggest that a cAMP signaling is defective inlearning-impaired aged rats (Bach, M. E. et al., Proc. Natl. Acad. Sci.USA, 96(9):5280-5285 (1999)).

[0035] The present invention relates to a novel methodology, alsoreferred to herein as augmented cognitive training (ACT), which can (1)rehabilitate various forms of cognitive dysfunction or (2) enhancenormal cognitive performance. ACT acts via a general molecular mechanismof synaptic plasticity, which apparently converts the biochemical effectof a newly acquired experience into a long-lasting structural change ofthe synapse. ACT can be applied for any aspect of brain function thatshows a lasting performance gain after cognitive training. Accordingly,ACT can be used in rehabilitating an animal with any form of cognitivedysfunction or in enhancing or improving any aspect of normal cognitiveperformance in an animal.

[0036] A growing body of evidence suggests that neurons continue toproliferate in the adult brain (Arsenijevic, Y. et al., Exp. Neurol.,170: 48-62 (2001); Vescovi, A. L. et al., Biomed. Pharmacother.,55:201-205 (2001); Cameron, H. A. and McKay, R. D., J. Comp. Neurol.,435:406-417 (2001); and Geuna, S. et al., Anat. Rec., 265:132-141(2001)) and that such proliferation is in response to variousexperiences (Nilsson, M. et al., J. Neurobiol., 39:569-578 (1999);Gould, E. et al., Trends Cogn. Sci., 3:186-192 (1999); Fuchs, E. andGould, E., Eur. J. Neurosci.,12: 2211-2214 (2000); Gould, E. et al.,Biol. Psychiatry, 48:715-720 (2000); and Gould, E. et al., Nat.Neurosci., 2:260-265 (1999)). Experimental strategies now are underwayto transplant neuronal stem into adult brain for various therapeuticindications (Kurimoto, Y. et al., Neurosci. Lett., 306:57-60 (2001);Singh, G., Neuropathology, 21:110-114 (2001); and Cameron, H. A. andMcKay, R. D., Nat. Neurosci., 2:894-897 (1999)). Much already is knownabout neurogenesis in embryonic stages of development (Saitoe, M. andTully, T., “Making connections between synaptic and behavioralplasticity in Drosophila”, In Toward a Theory of Neuroplasticity, J.McEachem and C. Shaw, Eds. (New York: Psychology Press.), pp. 193-220(2000)). Neuronal differentiation, neurite extension and initialsynaptic target recognition all appear to occur in anactivity-independent fashion. Subsequent synaptogenesis and synapticgrowth, however, then requires ongoing neuronal activity to fine-tunesynaptic connections in a functionally relevant manner. These findingssuggest that functional (final) integration of transplanted neural stemcells require neuronal activity. Thus, ACT can be used to exerciseappropriate neuronal circuits to fine-tune the synaptic connections ofnewly acquired, transplanted stem cells that differentiate into neurons.By “exercise appropriate neuronal circuit(s)” is meant the induction inthe appropriate neuronal circuit(s) of a pattern of neuronal activity,which corresponds to that produced by a particular cognitive trainingprotocol. The cognitive training protocol can be used to induce suchneuronal activity. Alternatively, neuronal activity can be induced bydirect electrical stimulation of the neuronal circuitry. “Neuronalactivity” and “neural activity” are used interchangeably herein.

[0037] ACT comprises a specific training protocol for each brainfunction and a general administration of CREB pathway-enhancing drugs.The training protocol (cognitive training) induces neuronal activity inspecific brain regions and produces improved performance of a specificbrain (cognitive) function. CREB pathway-enhancing drugs, also referredto herein as augmenting agents, enhance CREB pathway function, which isrequired to consolidate newly acquired information into LTM. By “enhanceCREB pathway function” is meant the ability to enhance or improveCREB-dependent gene expression. CREB-dependent gene expression can beenhanced or improved by increasing endogenous CREB production, forexample by directly or indirectly stimulating the endogenous gene toproduce increased amounts of CREB, or by increasing functional(biologically active) CREB. See, e.g., U.S. Pat. No. 5,929,223; U.S.Pat. No. 6,051,559; and International Publication No. WO9611270(published Apr. 18, 1996), which references are incorporated herein intheir entirety by reference. Administration of CREB pathway-enhancingdrugs decreases the training needed to yield a performance gain relativeto that yielded with training alone. In particular, ACT can enhancecognitive training by reducing the number of training sessions requiredto yield a performance gain relative to that yielded with cognitivetraining alone or by requiring shorter or no rest intervals betweentraining sessions to yield a performance gain. In this manner, ACT canimprove the efficiency of cognitive training techniques, therebyyielding significant economic benefit. By “performance gain” is meant animprovement in an aspect of cognitive performance.

[0038] The invention provides methods for enhancing a specific aspect ofcognitive performance in an animal (particularly in a human or othermammal or vertebrate) in need thereof comprising (a) administering tothe animal an augmenting agent which enhances CREB pathway function; and(b) training the animal under conditions sufficient to produce animprovement in performance of a particular cognitive task by the animal.

[0039] Training can comprise one or multiple training sessions and istraining appropriate to produce an improvement in performance of thecognitive task of interest. For example, if an improvement in languageacquisition is desired, training would focus on language acquisition. Ifan improvement in ability to learn to play a musical instrument isdesired, training would focus on learning to play the musicalinstrument. If an improvement in a particular motor skill is desired,training would focus on acquisition of the particular motor skill. Thespecific cognitive task of interest is matched with appropriatetraining.

[0040] The invention also provides methods for repeated stimulation ofneuronal activity or a pattern of neuronal activity, such as thatunderlying a specific neuronal circuit(s), in an animal comprising (a)administering to the animal an augmenting agent which enhances CREBpathway function; and (b) training the animal under conditionssufficient to stimulate or induce neuronal activity or a pattern ofneuronal activity in the animal. In this case, training is trainingappropriate to stimulate or induce neuronal activity or a pattern ofneuronal activity in the animal.

[0041] By “multiple training sessions” is meant two or more trainingsessions. The augmenting agent can be administered before, during orafter one or more of the training sessions. In a particular embodiment,the augmenting agent is administered before and during each trainingsession. Treatment with augmenting agent in connection with eachtraining session is also referred to as the “augmenting treatment”. By“training” is meant cognitive training.

[0042] Cognitive training protocols are known and readily available inthe art. See for example, Karni, A. and Sagi, D., “Where practice makesperfect in text discrimination: evidence for primary visual cortexplasticity”, Proc. Natl. Acad. Sci. USA, 88:4966-4970 (1991); Karni, A.and Sagi, D., “The time course of learning a visual skill”, Nature,365:250-252 (1993); Kramer, A. F. et al., “Task coordination and aging:explorations of executive control processes in the task switchingparadigm”, Acta Psychol. (Amst), 101:339-378 (1999); Kramer, A. F. etal., “Training for executive control: Task coordination strategies andaging”, In Aging and Skilled Performance: Advances In Theory andApplications, W. Rogers et al., eds. (Hillsdale, N.J.: Erlbaum) (1999);Rider, R. A. and Abdulahad, D. T., “Effects of massed versus distributedpractice on gross and fine motor proficiency of educable mentallyhandicapped adolescents”, Percept. Mot. Skills, 73:219-224 (1991);Willis, S. L. and Schaie, K. W., “Training the elderly on the abilityfactors of spatial orientation and inductive reasoning”, Psychol. Aging,1:239-247 (1986); Willis, S. L. and Nesselroade, C. S., “Long-termeffects of fluid ability training in old-old age”, Develop. Psychol.,26:905-910 (1990); Wek, S. R. and Husak, W. S., “Distributed and massedpractice effects on motor performance and learning of autisticchildren”, Percept. Mot. Skills, 68:107-113 (1989); Verhaehen, P. etal., “Improving memory performance in the aged through mnemonictraining: a meta-analytic study”, Psychol. Aging, 7:242-251 (1992);Verhaeghen, P. and Salthouse, T. A., “Meta-analyses of age-cognitionrelations in adulthood: estimates of linear and nonlinear age effectsand structural models”, Psychol. Bull., 122:231-249 (1997); Dean, C. M.et al., “Task-related circuit training improves performance of locomotortasks in chronic stroke: a randomized, controlled pilot trial”, Arch.Phys. Med. Rehabil., 81:409-417 (2000); Greener, J. et al., “Speech andlanguage therapy for aphasia following stroke”, Cochrane Database Syst.Rev., CD000425 (2000); Hummelsheim, H. and Eickhof, C., “Repetitivesensorimotor training for arm and hand in a patient with locked-insyndrome”, Scand. J. Rehabil. Med., 31:250-256 (1999); Johansson, B. B.,“Brain plasticity and stroke rehabilitation. The Willis lecture”,Stroke, 31:223-230 (2000); Ko Ko, C., “Effectiveness of rehabilitationfor multiple sclerosis”, Clin. Rehabil., 13 (Suppl. 1):33-41 (1999);Lange, G. et al., “Organizational strategy influence on visual memoryperformance after stroke: cortical/subcortical and left/right hemispherecontrasts”, Arch. Phys. Med. Rehabil., 81:89-94 (2000); Liepert, J. etal., “Treatment-induced cortical reorganization after stroke in humans”,Stroke, 31:1210-1216 (2000); Lotery, A. J. et al., “Correctable visualimpairment in stroke rehabilitation patients”, Age Ageing, 29:221-222(2000); Majid, M. J. et al., “Cognitive rehabilitation for memorydeficits following stroke”(Cochrane review), Cochrane Database Syst.Rev., CD002293 (2000); Merzenich, M. et al., “Cortical plasticityunderlying perceptual, motor, and cognitive skill development:implications for neurorehabilitation”, Cold Spring Harb. Symp. Quant.Biol., 61:1-8 (1996); Merzenich, M. M. et al., “Temporal processingdeficits of language-learning impaired children ameliorated bytraining”, Science, 271:77-81 (1996); Murphy, E., “Strokerehabilitation”, J. R. Coll. Physicians Lond., 33:466-468 (1999);Nagarajan, S. S. et al., “Speech modifications algorithms used fortraining language learning-impaired children”, IEEE Trans. Rehabil.Eng., 6:257-268. (1998); Oddone, E. et al., “Quality EnhancementResearch Initiative in stroke: prevention, treatment, andrehabilitation”, Med. Care 38:192-1104 (2000); Rice-Oxley, M. andTurner-Stokes, L., “Effectiveness of brain injury rehabilitation”, Clin.Rehabil., 13(Suppl 1):7-24 (1999); Tallal, P. et al., “Language learningimpairments: integrating basic science, technology, and remediation”,Exp. Brain Res., 123:210-219 (1998); Tallal, P. et al., “Languagecomprehension in language-learning impaired children improved withacoustically modified speech”, Science, 271:81-84 (1996); Wingfield, A.et al., “Regaining lost time: adult aging and the effect of timerestoration on recall of time-compressed speech”, Psychol. Aging,14:380-389 (1999), which references are incorporated herein in theirentirety by reference.

[0043] As used herein, the term “animal” includes mammals, as well asother animals, vertebrate and invertebrate (e.g., birds, fish, reptiles,insects (e.g., Drosophila species), mollusks (e.g., Aplysia). The terms“mammal” and “mammalian”, as used herein, refer to any vertebrateanimal, including monotremes, marsupials and placental, that suckletheir young and either give birth to living young (eutharian orplacental mammals) or are egg-laying (metatharian or nonplacentalmammals). Examples of mammalian species include humans and primates(e.g., monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs)and ruminents (e.g., cows, pigs, horses).

[0044] The animal can be an animal with some form and degree ofcognitive dysfunction or an animal with normal cognitive performance(i.e., an animal without any form of cognitive failure (dysfunction orloss of any cognitive function)).

[0045] Cognitive dysfunction, commonly associated with brain dysfunctionand central nervous system (CNS) disorders or conditions, arises due toheredity, disease, injury and/or age. CNS disorders and conditionsassociated with some form and degree of cognitive failure (dysfunction)include, but are not limited to the following:

[0046] 1) age-associated memory impairment;

[0047] 2) neurodegenerative disorders, such as delirium (acuteconfusional state); dementia, including Alzheimer's disease andnon-Alzheimer's type dementias, such as, but not limited to, Lewy bodydementia, vascular dementia, Binswanger's dementia (subcorticalarteriosclerotic encephalopathy), dementias associated with Parkinson'sdisease, progressive supranuclear palsy, Huntington's disease (chorea),Pick's disease, normal-pressure hydrocephalus, Creutzfeldt-Jakobdisease, Gerstmann-Sträussler-Scheinker disease, neurosyphilis (generalparesis) or HIV infection, frontal lobe dementia syndromes, dementiasassociated with head trauma, including dementia pugilistica, braintrauma, subdural hematoma, brain tumor, hypothyroidism, vitamin B₁₂deficiency, intracranial radiation; other neurodegenerative disorders;

[0048] 3) psychiatric disorders, including affective disorders (mooddisorders), such as, but not limited to, depression, includingdepressive pseudodementia; psychotic disorders, such as, but not limitedto, schizophrenia and autism (Kanner's Syndrome); neurotic disorders,such as, but not limited to, anxiety and obsessive-compulsive disorder;attention deficit disorder;

[0049] 4) trauma-dependent loss of cognitive function, such as, but notlimited to that associated with (due to), cerebrovascular diseases,including stroke and ischemia, including ischemic stroke; brain trauma,including subdural hematoma and brain tumor; head injury;

[0050] 5) disorders associated with some form and degree of cognitivedysfunction arising due to a genetic defect, such as, but not limitedto, Rubinstein-Taybi syndrome, down syndrome, Angelman syndrome, fragileX syndrome (fragile X-1, fragile X-2), neurofibromatosis, Coffin-Lowrysyndrome, myotonic dystrophy, Rett syndrome, William's syndrome,Klinefelter's syndrome, mosaicisms, trisomy 13 (Patau's syndrome),trisomy 18 (Edward's syndrome), Turner's syndrome, cri du chat syndrome,Lesch-Nyhan syndrome (hyperuricemia), Hunter's syndrome, Lowe'soculocerebrorenal syndrome, Gaucher's disease, Hurler's syndrome(mucopolysaccharidosis), Niemann-Pick disease, Tay-Sachs disease,galactosemia, maple syrup urine disease, phenylketonuria,aminoacidurias, acidemias, tuberous sclerosis and primary microcephaly;

[0051] 6) learning, language or reading disabilities, particularly inchildren. By “learning disabilities” is meant disorders of the basicpsychological processes that affect the way an individual learns.Learning disabilities can cause difficulties in listening, thinking,talking, reading, writing, spelling, arithmetic or combinations of anyof the foregoing. Learning disabilities include perceptual handicaps,dyslexia and developmental aphasia.

[0052] The terms “cognitive performance” and “cognitive function” areart-recognized terms and are used herein in accordance with theirart-accepted meanings. By “cognitive task” is meant a cognitivefunction. Cognitive functions include memory acquisition, visualdiscrimination, auditory discrimination, executive functioning, motorskill learning, abstract reasoning, spatial ability, speech and languageskills and language acquisition. By “enhance a specific aspect ofcognitive performance” is meant the ability to enhance or improve aspecific cognitive or brain function, such as, for example, theacquisition of memory or the performance of a learned task. By“improvement in performance of a particular cognitive task” is meant animprovement in performance of a specific cognitive task or aspect ofbrain function relative to performance prior to training. For example,if after a stroke, a patient can only wiggle his or her toe, animprovement in performance (performance gain) in the patient would bethe ability to walk, for example.

[0053] Accordingly, the invention also relates to methods of treating acognitive deficit associated with a CNS disorder or condition in ananimal (particularly in a human or other mammal or vertebrate) in needof said treatment comprising (a) administering to the animal anaugmenting agent which enhances CREB pathway function; and (b) trainingthe animal under conditions sufficient to produce an improvement inperformance of a particular cognitive task by the animal.

[0054] In one embodiment, the invention relates to a method of treatinga cognitive deficit associated with age-associated memory impairment inan animal in need of said treatment comprising (a) administering to theanimal an augmenting agent which enhances CREB pathway function; and (b)training the animal under conditions sufficient to produce animprovement in performance by the animal of a cognitive task whose lossis associated with age-associated memory impairment.

[0055] In a second embodiment, the invention relates to a method oftreating a cognitive deficit associated with a neurodegenerative disease(e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease,other senile dementia) in an animal in need of said treatment comprising(a) administering to the animal an augmenting agent which enhances CREBpathway function; and (b) training the animal under conditionssufficient to produce an improvement in performance by the animal of acognitive task whose deficit is associated with the neurodegenerativedisease.

[0056] In a third embodiment, the invention relates to a method oftreating a cognitive deficit associated with a psychiatric disease(e.g., depression, schizophrenia, autism, attention deficit disorder) inan animal in need of said treatment comprising (a) administering to theanimal an augmenting agent which enhances CREB pathway function; and (b)training the animal under conditions sufficient to produce animprovement in performance by the animal of a cognitive task whosedeficit is associated with the psychiatric disease.

[0057] In a fourth embodiment, the invention relates to a method oftreating a cognitive deficit associated with trauma dependent loss ofcognitive function (e.g., cerebrovascular diseases (e.g., stroke,ischemia), brain tumor, head or brain injury) in an animal in need ofsaid treatment comprising (a) administering to the animal an augmentingagent which enhances CREB pathway function; and (b) training the animalunder conditions sufficient to produce an improvement in performance bythe animal of a cognitive task whose deficit is associated with traumadependent loss of cognitive function.

[0058] In a fifth embodiment, the invention relates to a method oftreating a cognitive deficit associated with a genetic defect (e.g.,Rubinstein-Taybi syndrome, down syndrome, Angelman syndrome,neurofibromatosis, Coffin-Lowry syndrome, Rett syndrome, myotonicdystrophy, fragile X syndrome (e.g., fragile X-1, fragile X-2) andWilliam's syndrome) in an animal in need of said treatment comprising(a) administering to the animal an augmenting agent which enhances CREBpathway function; and (b) training the animal under conditionssufficient to produce an improvement in performance by the animal of acognitive task whose deficit is associated with a genetic defect.

[0059] In a particular embodiment, the invention relates to methods oftreating a cognitive deficit associated with mental retardation in ananimal in need of said treatment comprising (a) administering to theanimal an augmenting agent which enhances CREB pathway function; and (b)training the animal under conditions sufficient to produce animprovement in performance by the animal of a cognitive task whosedeficit is associated with mental retardation. In a particularembodiment, the augmenting agent is a phosphodiesterase 4 (PDE4)inhibitor. Examples of PDE4 inhibitors include rolipram and compounds ofthe following formula:

[0060] wherein “Me” means “methyl” and “cPent” means “cyclopentyl”. Itis understood that the above formula embraces both enantimers andmixtures thereof. The compounds can be prepared using the methodologyprovided in U.S. Pat. No. 6,458,829, the teachings of which areincorporated herein by reference. In a particular embodiment, the 3 and5 carbons of this above formula are in the S configuration:

[0061] wherein “Me” means “methyl” and “cPent” means “cyclopentyl”.Other examples of PDE4 inhibitors can be found in U.S. Publication No.2002/0028842 A1 (published Mar. 7, 2002); U.S. Pat. No. 6,458,829B1;U.S. Pat. No. 6,525,055B1; U.S. Pat. No. 5,552,438; U.S. Pat. No.6,436,965; and U.S. Pat. No. 6,204,275. Still other PDE4 inhibitors areknown and readily available in the art.

[0062] Mental retardation impacts cognitive processing and cognitivefunctions, including learning and memory acquisition (Weeber, E. J. etal., Neuron, 33:845-848)). Mental retardation may be caused bychromosomal or genetic factors, congenital infections, teratogens (drugsand other chemicals), malnutrition, radiation or unknown conditionsaffecting implantation and embryogenesis. Mental retardation syndromesinclude, but are not limited to, Klinefelter's syndrome, mosaicisms,trisomy 13 (Patau's syndrome), trisomy 18 (Edward's syndrome), Turner'ssyndrome, cri du chat syndrome, Lesch-Nyhan syndrome (hyperuricemia),Hunter's syndrome, Lowe's oculocerebrorenal syndrome, Gaucher's disease,Hurler's syndrome (mucopolysaccharidosis), Niemann-Pick disease,Tay-Sachs disease, galactosemia, maple syrup urine disease,phenylketonuria, aminoacidurias, acidemias, tuberous sclerosis andprimary microcephaly. Mental retardation syndromes also includeRubinstein-Taybi syndrome, down syndrome, Angelman syndrome,neurofibromatosis, Coffin-Lowry syndrome, Rett syndrome, myotonicdystrophy, fragile X syndrome (e.g., fragile X-1, fragile X-2) andWilliam's syndrome (Weeber, E. J. et al., Neuron, 33:845-848 (2002)).

[0063] The invention also relates to methods of therapy of a cognitivedeficit associated with a CNS disorder or condition in an animal havingundergone neuronal stem cell manipulation comprising (a) administeringto the animal an augmenting agent which enhances CREB pathway function;and (b) training the animal under conditions sufficient to stimulate orinduce neuronal activity or a pattern of neuronal activity in theanimal. By “neuronal stem cell manipulation” is meant that (1) exogenousneuronal stem cells are transplanted into the brain or spinal chord ofan animal or (2) endogenous neuronal stem cells are stimulated orinduced to proliferate in the animal. Methods of transplanting neuronalstem cells into the brain or spinal chord of an animal are known andreadily available in the art (see, e.g., Cameron, H. A. and McKay, R.D., Nat. Neurosci., 2:894-897 (1999); Kurimoto, Y. et al., Neurosci.Lett., 306:57-60 (2001); and Singh, G., Neuropathology, 21:110-114(2001)). Methods of stimulating or inducing proliferation of endogenousneuronal stem cells in an animal are known and readily available in theart (see, e.g., Gould, E. et al., Trends Cogn. Sci, 3:186-192 (1999);Gould, E. et al., Biol. Psychiatry, 48:715-20 (2000); Nilsson, M. et al,J. Neurobiol., 39:569-578 (1999); Fuchs, E. and Gould, E., Eur. JNeurosci., 12:2211-2214 (2000); and Gould, E. et al., Nat. Neurosci.,2:260-265 (1999)). The particular methods of transplanting neuronal stemcells into the brain or spinal chord of an animal and the particularmethods of stimulating or inducing proliferation of endogenous neuronalstem cells in an animal are not critical to the practice of theinvention.

[0064] The invention further relates to methods of improving orenhancing learning and/or performance in an animal with a learning,language or reading disability, or combinations of any of the foregoing,comprising (a) administering to the animal an augmenting agent whichenhances CREB pathway function; and (b) training the animal underconditions sufficient to produce an improvement in performance by theanimal of a cognitive task associated with the disability in learning,language or reading performance.

[0065] Augmenting agents, as used herein, are compounds withpharmacological activity and include drugs, chemical compounds, ioniccompounds, organic compounds, organic ligands, including cofactors,saccharides, recombinant and synthetic peptides, proteins, peptoids,nucleic acid sequences, including genes, nucleic acid products, andother molecules and compositions.

[0066] For example, augmenting agents can be cell permeant cAMP analogs(e.g, 8-bromo cAMP); activators of adenylate cyclase 1 (AC1) (e.g.,forskolin); agents affecting G-protein linked receptor, such as, but notlimited to adrenergic receptors and opioid receptors and their ligands(e.g., phenethylamines); modulators of intracellular calciumconcentration (e.g., thapsigargin, N-methyl-D-aspartate (NMDA) receptoragonists); inhibitors of the phosphodiesterases responsible for cAMPbreakdown (e.g., phosphodiesterase 1 (PDE1) inhibitors (e.g.,iso-buto-metho-xanthine (IBMX)), phosphodiesterase 2 (PDE2) inhibitors(e.g., iso-buto-metho-xanthine (IBMX)), phosphodiesterase 3 (PDE3)inhibitors, phosphodiesterase 4 (PDE4) inhibitors (e.g., rolipram,HT0712), etc.) (see also, e.g., U.S. Pat. No. 6,458,829B1; U.S.Publication No. 2002/0028842A1 (published Mar. 7, 2002)); and modulatorsof protein kinases and protein phosphatases, which mediate CREB proteinactivation and CREB-dependent gene expression. Augmenting agents can beexogenous CREB, CREB analogs, CREB-like molecules, biologically activeCREB fragments, CREB fusion proteins, nucleic acid sequences encodingexogenous CREB, CREB analogs, CREB-like molecules, biologically activeCREB fragments or CREB fusion proteins.

[0067] Augmenting agents can also be CREB function modulators, ornucleic acid sequences encoding CREB function modulators. CREB functionmodulators, as used herein, have the ability to modulate CREB pathwayfunction. By “modulate” is meant the ability to change (increase ordecrease) or alter CREB pathway function.

[0068] Augmenting agents can be compounds which are capable of enhancingCREB function in the CNS. Such compounds include, but are not limitedto, compounds which affect membrane stability and fluidity and specificimmunostimulation. In a particular embodiment, the augmenting agent iscapable of transiently enhancing CREB pathway function in the CNS.

[0069] CREB analogs, or derivatives, are defined herein as proteinshaving amino acid sequences analogous to endogenous CREB. Analogousamino acid sequences are defined herein to mean amino acid sequenceswith sufficient identity of amino acid sequence of endogenous CREB topossess the biological activity of endogenous CREB, but with one or more“silent” changes in the amino acid sequence. CREB analogs includemammalian CREM, mammalian ATF-1 and other CREB/CREM/ATF-1 subfamilymembers.

[0070] CREB-like molecule, as the term is used herein, refers to aprotein which functionally resembles (mimics) CREB. CREB-like moleculesneed not have amino acid sequences analogous to endogenous CREB.

[0071] Biologically active polypeptide fragments of CREB can includeonly a part of the full-length amino acid sequence of CREB, yet possessbiological activity. Such fragments can be produced by carboxyl or aminoterminal deletions, as well as internal deletions.

[0072] Fusion proteins comprise a CREB protein as described herein,referred to as a first moiety, linked to a second moiety not occurringin the CREB protein. The second moiety can be a single amino acid,peptide or polypeptide or other organic moiety, such as a carbohydrate,a lipid or an inorganic molecule.

[0073] Nucleic acid sequences are defined herein as heteropolymers ofnucleic acid molecules. The nucleic acid molecules can be doublestranded or single stranded and can be a deoxyribonucleotide (DNA)molecule, such as cDNA or genomic DNA, or a ribonucleotide (RNA)molecule. As such, the nucleic acid sequence can, for example, includeone or more exons, with or without, as appropriate, introns, as well asone or more suitable control sequences. In one example, the nucleic acidmolecule contains a single open reading frame which encodes a desirednucleic acid product. The nucleic acid sequence is “operably linked” toa suitable promoter.

[0074] A nucleic acid sequence encoding a desired CREB protein, CREBanalog (including CREM, ATF-1), CREB-like molecule, biologically activeCREB fragment, CREB fusion protein or CREB function modulator can beisolated from nature, modified from native sequences or manufactured denovo, as described in, for example, Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York (1998); and Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold SpringHarbor University Press, New York. (1989). Nucleic acids can be isolatedand fused together by methods known in the art, such as exploiting andmanufacturing compatible cloning or restriction sites.

[0075] Typically, the nucleic acid sequence will be a gene which encodesthe desired CREB protein, CREB analog, CREB-like molecule, CREB fusionprotein or CREB function modulator. Such a gene is typically operablylinked to suitable control sequences capable of effecting the expressionof the CREB protein or CREB function modulator, preferably in the CNS.The term “operably linked”, as used herein, is defined to mean that thegene (or the nucleic acid sequence) is linked to control sequences in amanner which allows expression of the gene (or the nucleic acidsequence). Generally, operably linked means contiguous.

[0076] Control sequences include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemessenger RNA (mRNA) ribosomal binding sites and sequences which controltermination of transcription and translation. In a particularembodiment, a recombinant gene (or a nucleic acid sequence) encoding aCREB protein, CREB analog, CREB-like molecule, biologically active CREBfragment, CREB fusion protein or CREB function modulator can be placedunder the regulatory control of a promoter which can be induced orrepressed, thereby offering a greater degree of control with respect tothe level of the product.

[0077] As used herein, the term “promoter” refers to a sequence of DNA,usually upstream (5′) of the coding region of a structural gene, whichcontrols the expression of the coding region by providing recognitionand binding sites for RNA polymerase and other factors which may berequired for initiation of transcription. Suitable promoters are wellknown in the art. Exemplary promoters include the SV40 and humanelongation factor (EFI). Other suitable promoters are readily availablein the art (see, e.g., Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, Inc., New York (1998); Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring HarborUniversity Press, New York (1989); and U.S. Pat. No. 5,681,735).

[0078] Augmenting agents can enhance CREB pathway function by a varietyof mechanisms. For example, an augmenting agent can affect a signaltransduction pathway which leads to induction of CREB-dependent geneexpression. Induction of CREB-dependent gene expression can be achieved,for example, via up-regulation of positive effectors of CREB functionand/or down-regulation of negative effectors of CREB function. Positiveeffectors of CREB function include adenylate cyclases and CREBactivators. Negative effectors of CREB function include cAMPphosphodiesterase (cAMP PDE) and CREB repressors.

[0079] An augmenting agent can enhance CREB pathway function by actingbiochemically upstream of or directly acting on an activator orrepressor form of a CREB protein and/or on a CREB protein containingtranscription complex. For example, CREB pathway function can beaffected by increasing CREB protein levels transcriptionally,post-transcriptionally, or both transcriptionally andpost-transcriptionally; by altering the affinity of CREB protein toother necessary components of the of the transcription complex, such as,for example, to CREB-binding protein (CBP protein); by altering theaffinity of a CREB protein containing transcription complex for DNA CREBresponsive elements in the promoter region; or by inducing eitherpassive or active immunity to CREB protein isoforms. The particularmechanism by which an augmenting agent enhances CREB pathway function isnot critical to the practice of the invention.

[0080] Augmenting agents can be administered directly to an animal in avariety of ways. In a preferred embodiment, augmenting agents areadministered systemically. Other routes of administration are generallyknown in the art and include intravenous including infusion and/or bolusinjection, intracerebroventricularly, intrathecal, parenteral, mucosal,implant, intraperitoneal, oral, intradermal, transdermal (e.g., in slowrelease polymers), intramuscular, subcutaneous, topical, epidural, etc.routes. Other suitable routes of administration can also be used, forexample, to achieve absorption through epithelial or mucocutaneouslinings. Particular augmenting agents can also be administered by genetherapy, wherein a DNA molecule encoding a particular therapeuticprotein or peptide is administered to the animal, e.g., via a vector,which causes the particular protein or peptide to be expressed andsecreted at therapeutic levels in vivo.

[0081] A vector, as the term is used herein, refers to a nucleic acidvector, e.g., a DNA plasmid, virus or other suitable replicon (e.g.,viral vector). Viral vectors include retrovirus, adenovirus, parvovirus(e.g., adeno-associated viruses), coronavirus, negative strand RNAviruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus(e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g.measles and Sendai), positive strand RNA viruses such as picornavirusand alphavirus, and double stranded DNA viruses including adenovirus,herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barrvirus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox andcanarypox). Other viruses include Norwalk virus, togavirus, flavivirus,reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.Examples of retroviruses include: avian leukosis-sarcoma, mammalianC-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus,spumavirus (Coffin, J. M., Retroviridae: The viruses and theirreplication, In Fundamental Virology, Third Edition, B. N. Fields, etal., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Otherexamples include murine leukemia viruses, murine sarcoma viruses, mousemammary tumor virus, bovine leukemia virus, feline leukemia virus,feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus,baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkeyvirus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcomavirus and lentiviruses. Other examples of vectors are described, forexample, in McVey et al., U.S. Pat. No. 5,801,030, the teachings ofwhich are incorporated herein by reference.

[0082] A nucleic acid sequence encoding a protein or peptide (e.g., CREBprotein, CREB analog (including CREM, ATF-1), CREB-like molecule,biologically active CREB fragment, CREB fusion protein, CREB functionmodulator) can be inserted into a nucleic acid vector according tomethods generally known in the art (see, e.g., Ausubel et al., Eds.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NewYork (1998); Sambrook et al., Eds., Molecular Cloning: A LaboratoryManual, 2nd edition, Cold Spring Harbor University Press, New York(1989)).

[0083] The mode of administration is preferably at the location of thetarget cells. In a particular embodiment, the mode of administration isto neurons.

[0084] Augmenting agents can be administered together with othercomponents of biologically active agents, such as pharmaceuticallyacceptable surfactants (e.g., glycerides), excipients (e.g., lactose),stabilizers, preservatives, humectants, emollients, antioxidants,carriers, diluents and vehicles. If desired, certain sweetening,flavoring and/or coloring agents can also be added.

[0085] Augmenting agents can be formulated as a solution, suspension,emulsion or lyophilized powder in association with a pharmaceuticallyacceptable parenteral vehicle. Examples of such vehicles are water,saline, Ringer's solution, isotonic sodium chloride solution, dextrosesolution, and 5% human serum albumin. Liposomes and nonaqueous vehiclessuch as fixed oils can also be used. The vehicle or lyophilized powdercan contain additives that maintain isotonicity (e.g., sodium chloride,mannitol) and chemical stability (e.g., buffers and preservatives). Theformulation can be sterilized by commonly used techniques. Suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences.

[0086] The dosage of augmenting agent administered to an animal is thatamount required to effect a change in CREB-dependent gene expression,particularly in neurons. The dosage administered to an animal, includingfrequency of administration, will vary depending upon a variety offactors, including pharmacodynamic characteristics of the particularaugmenting agent, mode and route of administration; size, age, sex,health, body weight and diet of the recipient; nature and extent ofsymptoms being treated or nature and extent of the cognitive function(s)being enhanced or modulated, kind of concurrent treatment, frequency oftreatment, and the effect desired.

[0087] Augmenting agents can be administered in single or divided doses(e.g., a series of doses separated by intervals of days, weeks ormonths), or in a sustained release form, depending upon factors such asnature and extent of symptoms, kind of concurrent treatment and theeffect desired. Other therapeutic regimens or agents can be used inconjunction with the present invention.

[0088] The present invention will now be illustrated by the followingexample, which is not to be considered limiting in any way.

EXAMPLE

[0089] This study was undertaken to demonstrate that a cognitive deficitassociated with mental retardation can be treated with aphosphodiesterase 4 (PDE4) inhibitor in conjunction with a cognitivetraining protocol. The study was conducted using an animal model ofRubinstein-Taybi syndrome (RTS).

[0090] RTS is a human genetic disorder characterized by mentalretardation and physical abnormalities including broad thumbs, big andbroad toes, short stature and craniofacial anomalies (Rubinstein, J. H.& Taybi, H., Am. J. Dis. Child., 105:588-608 (1963); Hennekam, R. C. etal., Am. J. Ment. Retard., 96:645-660 (1992); Levitas, A. S. & Reid, C.S., J. Intellect. Disabil. Res., 42(Pt 4):284-292 (1998); and Cantani,A. & Gagliesi, D., Eur. Rev. Med. Pharmacol. Sci., 2:81-87 (1998)). RTSoccurs in about 1 in 125,000 births and accounts for as many as 1 in 300cases of institutionalized mentally retarded people. In many patients,RTS has been mapped to chromosome 16p13.3 (Imaizumi, K. & Kuroki, Y.,Am. J. Med. Genet., 38:636-639 (1991); Breuning, M. H. et al., Am. J.Hum. Genet., 52:249-254 (1993); and Masuno, M. et al., Am. J. Med.Genet., 53:352-354 (1994)), the region of the gene encoding theCREB-binding protein (CBP) (Petrij, F. et al., Nature, 376:348-351(1995)). Many RTS patients are heterozygous for CBP mutations whichyield truncations of the CBP C-terminus, suggesting that adominant-negative mechanism may contribute to the clinical symptoms(Petrij, F. et al., Am. J. Med. Genet., 92:47-52 (2000)).

[0091] Mice carrying a truncated form of CBP show several developmentalabnormalities similar to patients with RTS. RTS patients suffer frommental retardation, while long-term memory formation is defective inmutant CBP mice. A critical role for cAMP signaling duringCREB-dependent long-term memory formation appears to be evolutionarilyconserved.

[0092] Methods

Mice

[0093] Generation of CBP^(±) mice was described by Oike et al. (HumanMolecular Genetics, 8:387-396 (1999)). CBP^(±) mice are an acceptedmouse model of Rubinstein-Taybi syndrome, particularly because (i) themolecular lesion (truncated protein) in CBP^(±) mice is similar to thoseknown for some RTS patients, (ii) CBP function in CBP^(±) heterozygousmice is reduced but not blocked and (iii) long-term memory formation,but not learning or short-term memory, appear specifically to bedisrupted in these mutant animals (Oike, Y. et al., Human MolecularGenetics, 8:387-396 (1999)). For these studies, animals were generatedby crossing CBP^(±) mice to C57BL/6 females (Jackson laboratory). Themice were genotyped with a PCR protocol as described previously (Oike,Y. et al., Human Molecular Genetics, 8:387-396 (1999)). Age-(12 to 14weeks old by the time of handling) and gender-matched mutant mice andwildtype littermates were used for all experiments.

[0094] The mice were kept on 12:12 light-dark cycle, and the experimentswere conducted during the light phase of the cycle. With the exceptionof training and testing times, the mice had ad lib access of food andwater. The experiments were conducted according to Animal WelfareAssurance #A3280-01 and animals were maintained in accordance with theAnimal Welfare Act and the Department of Health and Human Service guide.

Object Recognition Training and Testing

[0095] Mice were handled for 3-5 minutes for 5 days. The day beforetraining, an individual mouse was placed into a training apparatus (aPlexiglas box of L=48 cm; W=38 cm and H=20 cm, located intodimly-illuminated room) and allowed to habituate to an environment for15 minutes (see also Pittenger, C. et al., Neuron, 34:447-462 (2002)).Training was initiated twenty-four hours after habituation. A mouse wasplaced back into the training box, which contained two identical objects(e.g. a small conus-shape object), and allowed to explore these objects.The objects were placed into the central area of the box and the spatialposition of objects (left-right sides) was counterbalanced betweensubjects. Among experiments, training times varied from 3.5 to 20minutes.

[0096] Three separate, and otherwise experimentally naive, sets ofanimals were used. The first set was used for experiments summarized inFIGS. 3 and 4 (n=10 per genotype). The second set was used for theexperiment summarized in FIG. 5 (wildtype mice, n=20). The third set wasused for the experiment summarized in FIG. 6 (n=8 per genotype). Foreach experiment, the same set of animals was used repeatedly withdifferent (new) sets of objects for each repetition. All experimentswere designed and performed in a balanced fashion, meaning that: (i) foreach experimental condition (e.g. a specific dose-effect and/orgenotype-memory effect) 2-6 experimental mice and 2-6 control mice wereused; (ii) experiments with HT0712 injections consisted of thevehicle-injected mice and mice injected with 2-3 different doses ofHT0712; (iii) each experimental condition was replicated 2-4 independenttimes, and replicate days were added to generate final number ofsubjects.

[0097] Five-to-eight sessions were performed on each set of mice. Eachmouse was trained and tested no more than once per week and with aone-week interval between testing. In experiments with drug-injections(see below), vehicle-injected mice and high/low-dose-injected mice, werecounterbalanced. In each experiment, the experimenter was unaware(blind) to the treatment of the subjects during training and testing.

[0098] To test for memory retention, mice were observed for 10 minutes 3and 24 hours after training. Mice were presented with two objects, oneof which was used during training, and thus was ‘familiar’ and the otherof which was novel (e.g. a small pyramid-shape object). The test objectswere divided into ten sets of two “training” plus on “testing” objects,and a new set of objects was used for each training session. After eachexperimental subject, the apparatus and the objects were cleaned with90% ethanol, dried and ventilated for a few minutes.

Drug Compound Administration

[0099] Twenty minutes before training, mice were injected in their homecages with the indicated doses of HT0712 ((3S,5S)-5-(3-cyclopentyloxy-4-methoxy-phenyl)-3-(3-methyl-benzyl)-piperidin-2-one;also known as IPL 455,903)), Rolipram (in 1% DMSO/PBS) or with vehiclealone (1% DMSO/PBS). HT0712 has the following formula:

[0100] wherein “Me” means “methyl” and “cPent” means “cyclopentyl”.HT0712 can be prepared using the methodology provided in U.S. Pat. No.6,458,829B1, the teachings of which are incorporated herein byreference.

[0101] HT0712 was administered intraperitoneally (i.p.) at doses: 0.001mg/kg; 0.005 mg/kg; 0.01 mg/kg; 0.05 mg/kg; 0.1 mg/kg, 0.15 mg/kg and0.2 mg/kg. Rolipram (Sigma) was administered i.p. at dose 0.1 mg/kg.Drug compounds were injected with one week interval to allow sufficientwash-out time (the half-life for HT0712 and Rolipram<3 hours). Inaddition, vehicle- and drug-injected mice were counterbalanced fromexperiment to experiment. Such design allowed at least two weekswash-out time between repeated usages of high doses. Nodose-accumulating effects were observed with repeated injectionsbetween/within the groups.

Data Analysis

[0102] The experiments were videotaped via an overhead video camerasystem. Types were reviewed by a blinded observer and the followingbehavioral parameters were determined: time of exploration of an eachobject; the total time of exploration of the objects; number ofapproaches to the objects; and time (latency) to first approach to anobject. The discrimination index was determined as described previously(Ennaceur, A. & Aggleton, J. P., Behav. Brain Res., 88:181-193 (1997)).The data were analyzed by Student's unpaired t test using statisticalsoftware package (Statwiew 5.0.1; SAS Institute, Inc). All values in thetext and figure legends are expressed as mean±SEM.

[0103] Results

[0104] CBP is a transcriptional co-activator that binds tophosphorylated CREB (cAMP-response element binding protein)transcription factor to regulate gene expression (Lonze, B. E. & Ginty,D. D., Neuron, 35:605-623 (2002)). CREB-dependent gene expression hasbeen shown to underlie long-term memory formation in several vertebrateand invertebrate species (Poser, S. & Storm, D. R., Int. J. Dev.Neurosci., 19:387-394 (2001); Bailey, C. H. et al., Nat. Rev. Neurosci.,1:11-20 (2000); Dubnau, J. & Tully, T., Ann. Rev. Neurosci., 21:407-444(1998); and Menzel, R., Learn Mem., 8:53-62 (2001)), leading to theintriguing speculation that mental redardation in RTS patients mayderive from reduced CBP function during long-term memory formation(D'Arcangelo, G. & Curran, T., Nature, 376:292-293 (1995)). To this end,Oike et al. (Human Molecular Genetics, 8:387-396 (1999)) generated aC-terminal truncation mutation in mouse CBP, which appears to act in adominant-negative fashion to recapitulate many of the abnormalitiesobserved in RTS patients. Homozygous CBP^(−/−) mutants are embryoniclethal, while heterozygous CBP^(±) mice show reduced viability, growthretardation, retarded osseous maturation and hypoplastic maxilla (Oike,Y. et al., Human Molecular Genetics, 8:387-396 (1999)). Importantly,CBP^(±) A mice showed normal learning and short-term memory butdefective long-term memory for two passive avoidance tasks,substantiating the notion that normal CBP function is required formemory formation (Oike, Y. et al., Human Molecular Genetics, 8:387-396(1999)).

[0105] A high-throughput drug screen was accomplished using humanneuroblastoma cells, which were stably transfected with a luciferasereporter gene driven by a CRE (cAMP response element) promoter (a drugscreen for enhancers of CREB function) (Scott, R. et al., J. Mol.Neurosci., 19:171-177 (2002)). Cells were exposed to drug for two hoursand then stimulated with a suboptimal dose of forskolin for another fourhours. Compounds were selected that had no effect on their own but thatsignificantly increased forskolin-induced CRE-luciferase expression.Among the dozens of confirmed hits for several molecular targetsidentified from this screen, inhibitors of PDE4 were numerous.

[0106] As described herein, the PDE4 inhibitors HT0712 and rolipram,which has been shown previously to affect performance in animal modelsof memory (Barad, M. et al., Proc. Natl. Acad. Sci. USA, 95:15020-15025(1998); and Vitolo, O. V. et al., Proc. Natl. Acad. Sci. USA,99:13217-13221 (2002)), both produced robust effects on CRE-liciferaseexpression and on expression of a CRE-dependent endogenous gene,somatostatin (FIG. 2). Other PDE4 inhibitors are expected to producesimilar effects.

[0107] Initial experiments on normal, young adult mice established thatlong-term memory formation after contextual fear conditioning wasenhanced by PDE4 inhibitors (e.g., HT0712 and rolipram), delivered (i)directly to the hippocampus, (ii) intraventricularly or (iii)intraperitoneally (Scott, R. et al., J. Mol. Neurosci., 19:171-177(2002)). Specifically, these drugs enhanced memory formation by reducingthe amount of training required to produce maximal long-term memory.

[0108] To determine whether these drugs could ameliorate memory defectscaused by molecular lesions in the CREB pathway, the mouse model ofRubinstein-Taybi syndrome was used particularly because (i) themolecular lesion (truncated protein) in mice was similar to those knownfor some RTS patients, (ii) CBP function in CBP^(±) heterozygous micewas reduced but not blocked and (iii) long-term memory formation, butnot learning or short-term memory, appeared specifically to be disruptedin these mutant animals (Oike, Y. et al., Human Molecular Genetics,8:387-396 (1999)).

[0109] Long-term memory defects in CBP^(±) mutants have been reportedonly for fear-based tasks (Oike, Y. et al., Human Molecular Genetics,8:387-396 (1999)). Hence, it was first determined if CBP^(±) mutant micealso had defective long-term memory for a different type of task. Objectrecognition is a non-aversive task which relies on a mouse's naturalexploratory behavior. During training for this task, mice are presentedwith two identical novel objects, which they explore for some time byorienting toward, sniffing and crawling over. Mice then will rememberhaving explored that object. To test for such memory, mice are presentedat a later time with two different objects, one of which was presentedpreviously during training and thus is “familiar,” and the other ofwhich is novel. If the mouse remembers the familiar object, it spendsmore time exploring the novel object. By analogy to an objectrecognition-based ‘non-matching to sample’ task in monkeys and rats(Mishkin, M., Nature, 273:297-298 (1978); Mishkin, M. & Appenzeller, T.,Sci. Am., 256:80-89 (1987); and Wood, E. R. et al., Behav. Neurosci.,107:51-62 (1993)), this task can be performed repeatedly on the sameanimals by exposing them serially to different sets of novel objects.

[0110] Initially, CBP^(±) mutants and their wildtype (normal)littermates were given 15 minutes to explore a novel object duringtraining and then their memory retention was tested three and 24 hourslater (FIG. 3). Three-hour memory appeared normal, but 24-hour memorywas significantly reduced, in CBP^(±) mutants. These results indicatethat CBP^(±) mutant mice have impaired long-term memory, but normalshort-term memory, for object recognition. These findings extend theobservations of Oike et al. (Human Molecular Genetics, 8:387-396 (1999))to an ethologically relevant, non-aversive behavior and confirm thenotion that loss-of-function mutations in CBP can yield specific defectsin long-term memory formation.

[0111] To evaluate the PDE4 inhibitors, drug or vehicle alone wereadministered i.p. to normal mice and CBP^(±) mutants 20 minutes before a15-minute training session (FIG. 4). As in the previous experiment,24-hour memory retention was significantly reduced in CBP^(±) mutants inthe absence of drug. In striking contrast, however, a singleadministration of 0.10 mg/kg PDE4 inhibitor (e.g., HT0712 or rolipram)restored 24-hour memory in CBP^(±) mutants to normal levels.

[0112] To address whether the drugs' effects were specific to themolecular lesion in CBP, the training protocol was changed and dosesensitivity curves were determined for mutant and wild-type animals. The15-minute training protocol produces maximum 24-hour retention in thewildtype mice used here. Consequently, drug-induced memory enhancementin wildtype mice was not observed (FIG. 4). By reducing training to a3.5-minute protocol, 24-hour retention was near zero in wildtype mice,thereby allowing an evaluation of the enhancing effects of the PDE4inhibitors. Because CBP^(±) mutants had less functional CBP thanwildtype animals, a higher concentration of drug may be required in themutants than in wildtype mice to produce equivalent levels of memoryenhancement. In essence, the molecular lesion in CBP would act to shiftthe dose sensitivity for a PDE4 inhibitor to enhance memory formation.

[0113] Initially the dose-response curve for wildtype mice wasquantified (FIG. 5). In mice treated with vehicle alone, the 3.5-minutetraining protocol did not produce any appreciable 24-hour memory. Atconcentrations below 0.05 mg/kg or at 0.50 mg/kg, HT0712 failed toproduce any memory enhancement. Twenty-four hour memory retention wassignificantly increased, however, at concentrations of 0.05, 0.10, and0.15 mg/kg for HT0712. Next, memory retention was compared betweenCBP^(±) mutants and wildtype animals at selected concentrations ofHT0712 (FIG. 6). The initial effective dose was found to differ betweenmutant and wildtype animals. At a dose of 0.05 mg/kg for HT0712,wildtype animals showed significant enhancement of 24-hour memory, butCBP^(±) mutants did not. Memory enhancement was first seen in CBP^(±)mutants at the next higher dose of HT0712 (0.10 mg/kg). Similarly, thepeak effective dose appears shifted to a higher concentration in mutants(0.15 mg/kg) than in wildtype mice (0.10 mg/kg).

[0114] It was also considered whether HT0712 might be increasingperformance in the task nonspecifically by affecting perception of thetraining context (objects) or the motivation to explore objects duringtraining or testing. The latency to first approach an object duringtraining, the total number of approaches to an object and the totalexploration time were analyzed. In all experiments, no differencesbetween genotypes and/or drug treatments were observed in the latency tofirst approach. CBP^(±) mutant mice showed increases in totalexploration time and in the total number of object-approaches, but drugtreatments did not change these measures, and these behavioral responseswere not correlated with Discrimination Indices.

[0115] The data herein indicate that the memory impairments observed forCBP^(±) mutants in an object recognition task can be ameliorated byinhibitors of PDE4. These PDE inhibitors likely enhance signaling toCREB/CBP during memory formation by increasing cAMP levels in responseto experience-dependent changes in neural activity (Barad, M. et al.,Proc. Natl Acad. Sci. USA, 95:15020-15025 (1998); and Nagakura, A. etal., Neuroscience, 113:519-528 (2002)). Given the molecular andpathological similarities between these CBP^(±) mice and patients withRubinstein-Taybi syndrome, the findings herein indicate that PDE4inhibitors represent an effective therapy for the mental retardationassociated with this heritable condition by rescuing a functional defectin long-term memory formation and rendering the patient capable ofbenefitting from cognitive training and experience. The findings hereinalso imply that PDE4 inhibitors represent an effective therapy for othermental retardation syndromes, including Angelman syndrome,neurofibromatosis, Coffin-Lowry syndrome, down syndrome, Rett syndrome,myotonic dystrophy, fragile X syndrome (e.g., fragile X-1, fragile X-2)and William's syndrome, by treating a cognitive dysfunction or cognitivedeficit associated with the mental retardation and rendering the patientcapable of benefitting from cognitive training and experience.

[0116] All publications, patent and patent applications mentioned inthis specification are incorporated herein by reference to the sameextent as if each individual publication, patent or patent applicationwas specifically and individually incorporated by reference.

[0117] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of treating a cognitive deficitassociated with mental retardation in an animal in need of saidtreatment comprising the steps of: a) administering to said animal aphosphodiesterase 4 inhibitor; and b) training said animal underconditions sufficient to produce an improvement in performance by saidanimal of a cognitive task whose deficit is associated with mentalretardation, whereby said cognitive deficit is treated.
 2. The method ofclaim 1 wherein a performance gain is achieved relative to theperformance of said cognitive task achieved by training alone.
 3. Themethod of claim 1 wherein said mental retardation is associated with adisorder selected from the group consisting of: Rubinstein-Taybisyndrome, Down syndrome, Angelman syndrome, neurofibromatosis,Coffin-Lowry syndrome, Rett syndrome, myotonic dystrophy, fragile X-1syndrome, fragile X-2 syndrome and William's syndrome.
 4. The method ofclaim 1 wherein in step (b), training comprises multiple trainingsessions.
 5. The method of claim 1 wherein said phosphodiesterase 4inhibitor is administered before and during each training session. 6.The method of claim 1 wherein said phosphodiesterase 4 inhibitor isadministered after each training session.
 7. The method of claim 1wherein said animal is a mammal.
 8. The method of claim 7 wherein saidmammal is a human.
 9. A method of treating a cognitive deficitassociated with Rubinstein-Taybi syndrome in an animal in need of saidtreatment comprising the steps of: a) administering to said animal aphosphodiesterase 4 inhibitor; and b) training said animal underconditions sufficient to produce an improvement in performance by saidanimal of a cognitive task whose deficit is associated withRubinstein-Taybi syndrome, whereby said cognitive deficit is treated.10. The method of claim 9 wherein a performance gain is achievedrelative to the performance of said cognitive task achieved by trainingalone.
 11. The method of claim 9 wherein in step b), training comprisesmultiple training sessions.
 12. The method of claim 9 wherein saidphosphodiesterase 4 inhibitor is administered before and during eachtraining session.
 13. The method of claim 9 wherein saidphosphodiesterase 4 inhibitor is administered after each trainingsession.
 14. The method of claim 9 wherein said phosphodiesterase 4inhibitor is selected from the group consisting of: rolipram and HT0712.15. The method of claim 9 wherein said animal is a mammal.
 16. Themethod of claim 15 wherein said mammal is a human.